Infantile fiber-type I hypotrophy with simultaneously occurring severe onset of cardiomyopathy were previously reported in Dutch and Italian families and genetically linked to the MYL2 gene encoding for the human myosin regulatory light chain MLC2ventr/slow expressed in the ventricles and in slow-twitch skeletal muscles. Shortly after birth the patients experienced progressive slow-twitch skeletal myopathy and ultimately died of heart failure between 4 and 6 months of age. Dominant mutations in MYL2 have been known to cause familial hypertrophic cardiomyopathy (FHC) of extensive diversity in the course of the disease, age of onset and severity of symptoms. The mutation-specific dysregulation of the molecular events that trigger pathological remodeling of the heart, will be assessed using our transgenic (Tg) mice expressing the malignant: R58Q and D166V and benign: K104E mutations in MLC2ventr/slow. In addition to cardiac phenotypes, this application for the first time will include the slow-twitch skeletal muscle and the study of the splice site IVS6-1 mutation in MYL2 shown to cause severe myopathy in humans and premature death of IVS6-1-homozygous patients. AIM 1: Identify molecular mechanisms responsible for cardioskeletal dysfunction caused by MLC2ventr/slow mutations. We hypothesize that the mutation-induced structural changes trigger pathological remodeling of the heart and slow skeletal muscle leading to altered contractility and cardioskeletal myopathy. Proteomics study will be employed to identify the signaling pathways involved in cardioskeletal dysfunction associated FHC mutations. Structural phenotypes specific to MLC2ventr/slow mutations in the heart will be correlated with the respective phenotypes in the slow-twitch skeletal muscles using small angle X-ray diffraction patterns. Histopathology and electron microscopy (EM) will complement the effect of mutations on structural reorganization of the sarcomere in the heart and soleus muscle. Measurements of contractile force, force-pCa relationship and the myosin cross-bridge kinetics in skinned papillary/soleus muscle fibers from all proposed Tg mouse models of FHC will complete the phenotypic characterization of MLC2ventr/slow-specific cardioskeletal myopathy. Importantly, we will also study the IVS6-1 mutation associated with premature infantile cardiac death. AIM 2: Determine FHC induced cardiac phenotypes in vivo and explore novel rescue mechanisms in transgenic mice expressing constitutively phosphorylated P-MLC2. We hypothesize that by altering the Ca2+-dependent regulation of muscle contraction, D166V and R58Q mutations increase the propensity of affected patients toward malignant disease phenotypes. We also hypothesize that the underlying mechanisms relate to the steric inhibition of myosin light chain kinase dependent phosphorylation of MLC2. These hypotheses will be addressed using our recently developed double mutant Tg-S15D-D166V rescue mice, designed to mitigate the effects of the malignant D166V mutation with a constitutively phosphorylated Ser-15 (S15D).